Gene expression of the mouse corneal crystallin Aldh3a1:: Activation by Pax6, Oct1, and p300
Gene expression of the mouse corneal crystallin Aldh3a1:: Activation by Pax6, Oct1, and p300
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DOI：
10.1167/iovs.07-1057
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发表时间：
2008-05-01
影响因子：
4.4
通讯作者：
Piatigorsky, Joram
中科院分区：
文献类型：
--
作者：
Davis, Janine;Davis, Dionne;Piatigorsky, Joram
目的。醛脱氢酶3a1 （Aldh3a1）约占小鼠角膜上皮细胞水溶性蛋白的50%，与丰富的晶状体结晶蛋白相似，被认为是角膜结晶蛋白。本研究旨在研究小鼠角膜中Aldh3a1基因表达的发育模式和转录激活。定量(Q)-PCR和Western免疫印迹分析Aldh3a1 mRNA和蛋白表达。通过将含有与荧光素酶报告基因融合的Aldh3a1启动子可变部分的质粒共转染到具有选定转录因子的COS-7细胞中，检测功能启动子分析。转录因子结合位点通过电泳迁移转移法（EMSA）和染色质免疫沉淀法（ChIP）进行鉴定。采用原位杂交和免疫组织化学方法检测Aldh3a1、Pax6和Oct1在角膜中的表达。Aldh3a1的表达从出生时开始在角膜中暂时受到调节，到6周龄时增加100倍。Pax6、Oct1和p300协同激活Aldh3a1启动子的频率为116倍。在Aldh3a1启动子片段中分别鉴定出1个Pax6和2个Oct1结合位点。Pax6和Oct1均存在于6周龄小鼠角膜上皮细胞的细胞核中。最后，杂合子小眼Pax6+/-小鼠角膜中Aldh3a1的减少与Pax6的减少相关。Pax6、Oct1和p300激活小鼠角膜结晶蛋白Aldh3a1的基因表达。这些转录因子也与晶状体中晶体蛋白基因的高表达有关，这与“折射假说”一致，“折射假说”将晶状体和角膜的许多方面统一起来。
PURPOSE. Aldehyde dehydrogenase 3a1 (Aldh3a1) represents similar to 50% of the water-soluble protein of the mouse corneal epithelial cells and thus, by analogy with the abundant lens crystallins, is considered a corneal crystallin. This study was conducted to examine the developmental pattern and transcriptional activation of Aldh3a1 gene expression in the mouse cornea.METHODS. Aldh3a1 mRNA and protein were analyzed by quantitative (Q)-PCR and Western immunoblot analysis. Functional promoter analysis was examined by cotransfecting plasmids containing variable portions of the Aldh3a1 promoter fused to the luciferase reporter gene into COS-7 cells with selected transcription factors. Transcription factor binding sites were identified by electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation assays (ChIP). In situ hybridization and immunohistochemistry were used to assess expression of Aldh3a1, Pax6, and Oct1 in the cornea.RESULTS. Aldh3a1 expression is temporally regulated in the cornea beginning at birth and increasing 100-fold by 6 weeks of age. Pax6, Oct1, and p300 synergistically activate the Aldh3a1 promoter similar to 116-fold. One Pax6 and two Oct1 binding sites were identified in vitro and in vivo in the Aldh3a1 promoter fragment analyzed. Pax6 and Oct1 are both present in the nuclei of corneal epithelial cells of the 6-week-old mouse. Finally, a reduction of Aldh3a1 correlated with reduced Pax6 in the corneas of heterozygous Small eye Pax6+/- mice.CONCLUSIONS. Pax6, Oct1, and p300 activate gene expression of the corneal crystallin Aldh3a1 in the mouse. These transcription factors are also implicated in the high expression of crystallin genes in the lens, consistent with the "refracton hypothesis" unifying many aspects of the lens and cornea.